Abstract : The development of simple molecular assays with membrane protein receptors in a native conformation still represents a challenging task. Exosomes are extracellular vesicles which, due to their stability and small size, are suited for analysis in various assay formats. Here, we describe a novel approach to sort recombinant fully native and functional membrane proteins to exosomes using a targeting peptide. Specific binding of high affinity ligands to the potassium channel Kv1.2, the G-protein coupled receptor CXCR4, and the botulinum neurotoxin type B (BoNT/B) receptor, indicated their correct assembly and outside out orientation in exosomes. We then developed, using a label-free optical biosensor, a new method to determine the kinetic constants of BoNT/B holotoxin binding to its receptor synaptotagmin2/GT1b ganglioside (k on = 2.3 ×10 5 M −1 .s −1 , k off = 1.3 10 −4 s −1), yielding an affinity constant (K D = 0.6 nM) similar to values determined from native tissue. In addition, the recombinant binding domain of BoNT/B, a potential vector for neuronal delivery, bound quasi-irreversibly to synaptotagmin 2/GT1b exosomes. Engineered exosomes provide thus a novel means to study membrane proteins for biotechnology and clinical applications. Determination of the binding kinetics of analytes is potentially extremely useful for drug development and assay validation. In principle, purified endogenous or recombinant membrane proteins can be integrated into a lipid environment, such as proteoliposomes, supported lipid bilayers or nanodiscs. However, this approach is not ideal, due to the fact that membrane proteins are difficult to produce, prone to denaturation and do not always retain their functional integrity in artificial lipid bilayers 1, 2. Exosomes are extracellular vesicles, produced by many cell types and can be detected or purified using anti-bodies 3, 4. Furthermore, the ability of exosomes to carry molecules from native or engineered parental cells and fuse with recipient cells to deliver their cargo, confers considerable biomedical potential 5, 6. Unlike intra-cellular vesicles, exosomes display homogeneous topology in which plasma membrane proteins have the same outside-out orientation as at the surface of intact cells. Moreover, exosomes can potentially provide the means of expressing recombinant proteins in small membrane vesicles that can be used as vaccine strategies 7, 8. Our principal aim was to develop a general approach to address recombinant membrane proteins to exosomes, using a specific targeting sequence to direct exosomal sorting of a set of membrane drug targets, with a particular focus on the botulinum neurotoxin/B (BoNT/B) receptor. BoNT serotypes A, B and E (BoNT/A, BoNT/B, BoNT/E), produced by the bacteria Clostridium botulinum are the main cause of human botulism and are produced as a complex of holotoxin (MW 150 000 Da) and several nontoxic proteins. Although BoNTs are among the most neurotoxic substances known, their ability to reversibly block cholinergic nerves has provided the basis 1 INSERM